A map of glycation and glycoxidation sites in collagen I of human cortical bone: Effects of sex and type 2 diabetes

Abstract

Complications of diabetes is a major health problem affecting multiple organs including bone, where the chronic disease increases the risk of fragility fractures. One hypothesis suggests a pathogenic role for hyperglycemia-induced modification of proteins, a.k.a. advanced glycation end products (AGEs), resulting in structural and functional damage to bone extracellular matrix (ECM). Evidence supporting this hypothesis has been limited by the lack of comprehensive information about the location of AGEs that accumulate in vivo at specific sites within the proteins of bone ECM. Analyzing extracts from cortical bone of cadaveric femurs by liquid chromatography tandem mass spectrometry, we generated a quantitative AGE map of human collagen I for male and female adult donors with and without diabetes. The map describes the chemical nature, sequence position, and levels of four major physiological AGEs, e.g. carboxymethyllysine, and an AGE precursor fructosyllysine within the collagen I triple-helical region. The important features of the map are: 1) high map reproducibility in the individual bone extracts, i.e. 20 male and 20 female donors; 2) localization of modifications to distinct clusters: 10 clusters containing 34 AGE sites in male donors and 9 clusters containing 28 sites in female donors; 3) significant increases in modification levels in diabetes at multiple sites: 26 out of 34 sites in males and in 17 out of 28 sites in females; and 4) generally higher modification levels in male vs. female donors. Moreover, the AGE levels at multiple individual sites correlated with total bone pentosidine levels in male but not in female donors. Molecular dynamics simulations and molecular modeling predicted significant impact of modifications on solvent exposure, charge distribution, and hydrophobicity of the triple helix as well as disruptions to the structure of collagen I fibril. In summary, the AGE map of collagen I revealed diabetes-induced, sex-specific non-enzymatic modifications at distinct triple helical sites that can disrupt collagen structure, thus proposing a specific mechanism of AGE contribution to diabetic complications in human bone.